A vast array of proteins use molecular oxygen to carry out biotransformations within the cell. The existing O2 in the environment is a ground state triplet and, generally, must undergo activation to a partially reduced species before adding organic substrates. A major challenge is to understand how proteins achieve the activation of O2 without incurring oxidative damage to themselves and/or other cellular components. This laboratory has developed a suite of generic experimental probes that is applicable to a wide range of proteins that act on molecular oxygen. These probes allow us to address the nature of O2 binding to protein, whether electron and/or proton transfer to O2 is rate determining, and the properties of reactive intermediates that may accumulate during catalytic turnover. Representative examples of proteins from major classes of O2 activating enzymes have been chosen for study. These include (i) glucose oxidase, as a paradigmatic FAD-containing protein; (ii) lipoxygenase from soybean, as a model for the mammalian enzyme and a representative of a mononuclear iron center that uses metal to activate substrate; (iii) 1-aminocyclopropane carboxylate (ACC) oxidase, a 2-His-1-Carboxylate mononuclear iron protein that uses metal to activate O2; (iv) cytochrome P450, a heme iron enzyme; (v) peptidylglycine alpha-hydroxylating monooxygenase (PHM) and dopamine beta-monooxygenase (DBetaM), belonging to a singular class of eukaryotic proteins with two copper centers at a distance of ca. 10-11 Angstroms; and (vi) the copper amine oxidases (CAOs), containing TPQ and copper at their active sites. The above described enzymes catalyze reactions of significant physiological importance, for example, the first committed step in leukotriene biosynthesis (mammalian lipoxygenase), the production of neuroactive hormones and transmitters in mammals and plants (PHM, DBetaM, and ACC oxidase), the metabolism of xenobiotics (P450), and the production of hydrogen peroxide as a putative extra-cellular signaling agent in mammalian cells (CAOs). With the exception of ACC oxidase, each of the described enzyme systems catalyzes C-H cleavage reactions, and the details of the hydrogen transfer processes are under investigation in concert with studies of O2 reactivity. A major objective of this proposal is to move beyond the particulars of a single enzyme system, to generate fundamental understanding of the principles that govern biological O2 and C-H activation.